Gene Modified T CellsOncology Immunotherapeutics-I

Laurence J.N. Cooperljncooper@mdanderson.org

10-23-201410:30 am to 12:00 pm

Disclosure. Dr. Laurence J. N. Cooper has been a consultant for American Stem Cells, Inc., GE Healthcare, Ferring Pharmaceuticals, Inc., and Bristol-Myers Squibb. Dr. Cooper has received multiple grants from foundations in the state of Texas, including CPRIT and UT STAR, and Federal to support research. Dr. Cooper has received honoraria and payment for the development of education presentations including service on speakers’ bureaus from Miltenyi Biotec. Dr. Cooper has received travel/accommodations expenses covered/reimbursed by Lonza.

Thanks to Dr. George McNamara

Broadening the clinical appeal of genetically modified T cells

Torikai H, Moyes JS, Cooper LJN 2014 engineering T cells to target tumor cells. In W. Cai (ed) Engineering in Translational Medicine, Springer-Verlag London 2014.

Tumor-associated antigens targeted by CARs

Identify tumor-associated antigens

Collaboration with Immatics and XPRESIDENT® Discovery Platform

LB Alexandrov et al. Nature, 1-7 (2013) doi:10.1038/nature12477

Requires targeting self antigens? Targeting neoantigens possible?

• Boundaries on this slide are arbitrary• Immunogenic neoantigens may be identified by exome sequencing with sufficient probability only on

mutation-rich tumors• Identifying immunogenic self antigens will require a different technology, e.g. mass spectrometry

applicable to all HLA expressing tumors

Targeting tumor-associated antigens

Broadening the clinical appeal of genetically modified T cells

GOF Gain of functionLOF Loss of functionDGF Dominant gain of functionDNF Dominant negative form

Approaches to gene transfer

Viral• Retrovirus • Lentivirus

• Enhancer deleted, self inactivating (SIN) long terminal repeat lentivirus vectors. • Adenovirus• AAV• other

Non-viral • DNA (electroporation or other)• Sleeping Beauty (SB11, SB100X)• piggyBac

mRNA• Transient production• Artificial nucleases, transposases, recombinases

TransposasemRNA

Nucleus

Transposon

CAR

Cytoplasm

Genetic modification of T cells to redirect specificity

Kershaw, Westwood, Darcy Nature Reviews Cancer 13, 525–541 (2013)

Personalizing the therapy:Manipulating the T cell by harvesting and re-expressing melanoma-

specific TCR

6 OCTOBER 2006 VOL 314 SCIENCE

TCR gene therapy in patients with metastatic melanoma

J Clin Oncol 2011;29:917-9243 (3, 8, 9+)2 (22+, 20+)511humanNYESO1/A2

Blood. 2009;114:535-5462 (4, 3)1 (14+)316mousegp100/A2

Blood. 2009;114:535-5466 (3, 4, 9, 16+, 17+, 17+)

620human high avidityMART-1/A2

Science 2006; 314:126-129 Blood. 2009;114:535-5464431humanMART-1/A2

ReferencePRCRResponsePatient TCRabTarget Antigen

Torikai, Moyes, Cooper 2014 Engineering T cells to target tumor cells. In W. Cai (ed) Engineering in Translational Medicine, Springer-Verlag London 2014.

Govers et al 2014 J Immunol

CD3ε (epsilon)

not CD3ζ (zeta)

Avoiding TCR miss-pairing

Zhang, Morgan Adv Drug Delivery Rev 2012 64: 756–762.Provasi et al., Nat Med. 2012 May;18(5):807-15. Torikai et al., Blood. 2012 Jun 14;119(24):5697-705.

Identification of target antigens and TCRs for CD8+ T cells

Siewert et al., Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides. Nat Med. 2012.

Outcomes and toxicities infusing CD19-specific CAR+ T cells

NucleasesCRISPR/Cas9MeganucleasesTALENsZFNs

Recombinases / TransposasesAdenovirusCreSB11, SB100X piggyBac

ActivationsgRNA:dCas9-TFTALEZFP-TF

site specific integration by HR, mutations by NHEJ.clustered regularly interspaced short palindromic repeat (RNA)/ Cas9I-CreI meganucleases derived from self-splicing introns/inteinsTAL Effector NucleasesZinc Finger Nucleases

Adenovirus delivery of TALENs+DNA with protein capped ends (Holkers 2014)Cre recombinaseSleeping Beauty transposase (2 of various versions), random AT sitespiggyBac transposases (various versions), random AATT sites

Synthetic guide RNA:disabled Cas9 nuclease-Transcription FactorTranscription Activator like-EffectorZinc Finger Protein-Transcription Factor

Alternative functional domains include: Paired Nickases (increase selectivity for double stranded breaks), recombinases, repressors, epigenomic modifiers (DNA methyltransferases, histone acetylases or deacetylases, etc), fluorescent proteins (SUNtags, TALEcolor, TALE-Lights).NHEJ: Non-homologous end joining (& micro-homologous recombination).HR: Homologous recombination.Select companies in this field are highlighted later.

Approaches to gene editing, genome engineering

Artificial nucleases

http://www.toolgen.com/html/eng/technology/engineered_nucleases.php

Hard

Easy

Clinical

Research

Adenosine

A2AR (drug or delete gene)

BCM: “Rewire”: TGFβRex-TLR4in co-stimulationArginaseand iNOSdeplete Arg, iNOS generatesH2O2

TILs, CAR or Delete

HypoxiaHigh adenosine, H2O2, lactate, PGE2, TGFβArg, Trp depletion

Tumor

Adapted from: Gattinoni, Klebanoff, Restifo 2012 Nat Rev Cancer 12: 671-684

Cellular substrates

CAR designscFv • affinity • Other binders … ex. D-CAR based on Dectin-1

(Kumarasen et al. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):10660-5. )

Stalk• Which stalk … Ig vs. CD8 vs. short• Length of hinge+ stalk for proximal vs. distal

epitopes• Reduce FcR bindingSignaling domains• Signal 1, 2, 3

Jena et al. Curr Hematol Malig Rep. 2014 Mar;9(1):50-6.

CAR designs

2nd: inhibitory costimulation modulatory ligands-> signal(s) K = kinase docking sites P = phosphorylation sites Other sites possible (ITAMs, ITIMS …)Abate-Daga, et al., Oncoimmunology. 2014 Jun 18;3:e29194.

Combinatorial CARs

Conditional ablation of T cells

Casucci, Bondanza 2011 J of Cancer

HSV-tk: phosphorylates pro-drug ganciclovir, that is then incorporated into DNA.HSV-tk can be immunogenic

• CD3 with CD28 beads (Novartis)• CD3 and CD28 beads (TransACT, Miltenyi Biotec)• Activating and propagating cells (AaPC),

• many variations … different outputs

Forget et al. 2014 J Immunother 37: 448-460.

Approaches to propagation of genetically modified T cells

Huls et al., J Vis Exp. 2013 Feb 1;(72):e50070. doi: 10.3791/50070.

Gattinoni, Klebanoff, Restifo 2012 Nat Rev Cancer 12: 671-684.

T-cell subpopulations for genetic engineering

Lymphocyte populations for engineering

* mbIL-21 AaPC

CD16 T cells Deniger, … Cooper 2014 Clin Cancer Res

T cells Kudo, … Campana 2014 Cancer ResNK cells Chu, ... Yu, Hofmeister 2014 Leukemia NK cells* Denman, ... Cooper, Lee 2012 PLoS OneNKT cells Heczey, ... Metelitsa 2014 Blood

Broadening the clinical appeal of genetically modified T cells

Approaches to manufacture and distribution of engineered T cells

T cells are rendered as a drug

Insert CAR or TCR

CAR

Patient

CD19

Intended response

B-cell leukemia/lymphomaHLAs

TCRαβ

Normal cells

HLAs

Unwanted response

Recognize “non-self” (Donor →Patient)

GVHD

Gene insertion:Retrovirus/lentivirus

Sleeping BeautymRNA

Artificial nuclease

Eliminating TCR on CAR+ T cells

Blood. 2013 Aug 22;122(8):1341-9Blood. 2012 Jun 14;119(24):5697-705 Cellectis/Pfizer

Summary

Inter- and intra-tumor heterogeneity

Infuse T cells with one or more specificityPersonalized for the patient and the disease

“N=1” trial paradigm 27

T cells are precision tools

CAR+ T cells

CAR+ T cells

CAR+ T cells

CAR+ T cells

CAR+ T cells

TCR+ T cells

TCR+ T cells

TCR+ T cells

TCR+ T cells

TCR+ T cells

Power-law curveThe teaching of Beyoncé

Cost of distribution

Num

ber o

f pur

chas

es

Number of artists

Brick & Mortar

iTunes

Power-law curveThe new industrialization of T cells

Cost of distribution

Number of trials

Num

ber o

f pati

ents

Traditional Med Centers

Immuno-oncology at Med Centers1

The N=1 approach has been shown for non-genetically modified T cells

Evidence of tumor regression after treatment with a highly pure population of V22+

ERBB2IP mutation–reactive CD4+ T cells.

COMMERCIALIZATION OF T-CELL THERAPIES

Cell Immunotherapy of Cancer – Deals 2012-2014

LC 10/22/2014. Data from Sheridan 2013, 2014 Nat Biotechnol; Herper 2014 Forbes; MDA Strategic Alliances web page; Companies Internet web sites; current news.

Institution Amount Year Emphasis

Adaptimmune $104M 2014 Series A financing, enhanced TCR(s) to cancer specific antigens (NY-ESO-1, …)

Amgen-Micromet $1.16B 2012 Acquisition. BiTES, including Blinatumomab (CD3&CD19 bispecific antibody)

Argos Therapeutics $42M 2013 Dendritic cell personalized kidney cancer vaccine AGS-003

AstroZeneca/MedImmune-Amplimmune $225M* 2013 PD-1 inhibitor AMP-514; preclinical B7 pathway molecules. * up to $275M milestones.

AstroZeneca/MedImmune-MDA 2014 Research – Moon Shot Program’s Immunotherapy platform

Bellicum Pharmaceuticals $69M* 2014 CAR T-cells, iCasp-9 inducible suicide gene switch (2014 series C $55M; 2014 series B $14M)

Bellicum Pharmaceuticals-Ariad $50M 2014 Bellicum worldwide exclusive license to Dimerizer (ex. AP1903) for use in human cell therapies for all diseases.

Bluebird Bio-Celgene $225M* 2013 Chimeric antigen receptor (CAR) T-cells; *up to $225M per product

Bristol-Myers Squibb-Five Prime Ther. $50M* 2014 Two immune checkpoint pathways. * up to $300M milestones

Bristol-Myers Squibb 2014 Nivolumab (anti-PD-1) FDA approval (Ipilimumab, anti-CTLA, was approved in 2011)

Cell Medica $15 2012 CPRIT Commercialization award. EBV+ tumor cells antigen specific T-cells in collaboration with BCM.

GlaxoSmithKline-MDA $335M* 2012 OX40L mimicking mAbs (co-stimulation)

Johnson&Johnson/Janssen Biotech-MDA 2014 Research – Moon Shot Program’s Immunotherapy platform

Jounce Therapeutics $47M 2013 Series A funding. Founders: Allison, Gajewski, Pardoll, Sharma, Weiner

Juno Therapeutics $300M 2013 Chimeric antigen receptor (CAR) T-cells (MSKCC, FHCRC CAR T-cells)

Lion Biotechnologies $23M 2013 Tumor infiltrating lymphocytes (TILs, Rosenberg-NCI). SAB: Hwu, Radvanyi, Yee.

Kite Pharma-NCI/NIH $128M 2014 Rosenberg CAR T-cell therapies (CART’s)

Merck-Ablynx $20M* 2014 Immune checkpoint targeting nanobodies. * plus up to $2.3B royalties

Merck-Pfizer 2014 Collaboration: PD-1 inhibitor pembrolizumab (MK-3475); TKI Inlyta; 4-1BB agonist

Merck-Incyte 2014 Collaboration: PD-1 inhibitor pembrolizumab (MK-3475) plus INCB24360 IDO inhibitor

Novartis-CoStim 2014 Acquisition. Co-stimulatory pathways pipeline

Novartis-Dendreon $43M 2012 Novartis bought cell manufacturing plant (i.e. for UPenn CAR T-cells)

Novartis-UPenn $20M* 2012 Chimeric antigen receptor (CAR) T-cells. * plus milestones and royalties.

Pfizer-Cellectis $110M 2014 CAR T-cells, TALEN genome engineering. * plus up to $2.9B milestones

Pfizer-MDA 2014 Research – Moon Shot Program’s Immunotherapy platform

Pierre Fabre-Aurigene 2014 AUNP-12, 29 amino acid peptide based PD-1 inhibitor (license)

Roche-Immatics $17M* 2013 Access to Immatics proprietary TUMAP peptide vaccines. * plus up to $1B research funding and milestones.

Roche-Inovia $10M* 2013 Two of Inovia’s preclinical therapies. * Plus up to $412M in funding and potential milestones.

UNUM Therapeutics $12M 2014 Series A funding. Founder: Dario Campana. T-cells with CD16 FcReceptors to bind mAbs.